WHO grade IV malignant glioma is emblematic for a majority of cancers, where the efficacy of immune checkpoint blockade is impeded by a low mutational burden and notorious immunosuppression incited by a non-engaged tumor microenvironment. To overcome these hurdles and achieve successful immunotherapy of malignant glioma (or other `non-immunogenic' cancers), novel approaches must be capable of immunologically engaging the tumor microenvironment (TME) to instigate new antitumor immune repertoire. Our project is focused on the polio:rhinovirus chimera PVSRIPO, and its capacity to elicit inflammatory responses in the TME. PVSRIPO yields promising responses after single, convection-enhanced intratumoral infusion in patients with recurrent WHO grade IV malignant glioma. It targets neoplastic cells for infection and destruction by virtue of ectopic expression of its receptor, the CD155 tumor antigen, on such cells. We hypothesize, however, that the key to PVSRIPO's immunotherapy potential is poliovirus' inherent tropism for antigen presenting cells (APCs), e.g. tumor-associated macrophages, and a peculiar resistance to the innate antiviral interferon (IFN) response. PVSRIPO infection of APCs elicits sublethal viral propagation that provokes profound, sustained type I IFN responses and stimulates T cell co-stimulating functions. There is broad agreement that the unparalleled stimulus of the innate antiviral response in APCs is pivotal for generating tumor antigen-specific antitumor immunity. As with the role of APCs in natural poliovirus infection, the nature and extent of PVSRIPO interactions with myeloid host cells in tumors in vivo are unknown. For example, the mechanisms permitting chronic PVSRIPO replication with subdued cytotoxicity in APCs -necessary preludes to their IFN-dominant activation- remain obscure. The investigations proposed in this project are of utmost importance for the clinical development of PVSRIPO immunotherapy in malignant gliomas and beyond. Therefore, we are pursuing the following Specific Aims: 1) Define the molecular basis for PVSRIPO translation competency, cytotoxicity and type I IFN induction in macrophages/myeloid cells. We will perform studies in human myeloid-derived macrophages to mechanistically decipher the unique relationship of PVSRIPO with APCs; 2) Unravel PVSRIPO's potential for reprogramming macrophages/myeloid cells in the glioma TME in vivo and determine the role of macrophages in PVSRIPO immunotherapy. We will use transplantable chemically-induced- and genetically-engineered immunocompetent mouse glioma models to investigate the role of tumor stroma in PVSRIPO-instigated antitumor immunity; 3) Elucidate PVSRIPO targeting of macrophages/myeloid cells in primary human glioblastoma explants and examine proinflammatory reprogramming in vitro. We will decipher the TME response to PVSRIPO infection in fresh, non-dissociated primary tissue explants from glioma patients and measure its potential in overcoming T cell suppression.